Wheat plants having increased resistance to imidazolinone herbicides

ABSTRACT

The present invention is directed to wheat plants having increased resistance to an imidazolinone herbicide. More particularly, the present invention includes wheat plants containing one or more IMI nucleic acids such as a Teal IMI cultivar. The nucleic acids are preferably located on or derived from different genomes. The present invention also includes seeds produced by these wheat plants and methods of controlling weeds in the vicinity of these wheat plants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. ProvisionalApplication Ser. No. 60/311,282 filed Aug. 9, 2001.

FIELD OF THE INVENTION

The present invention relates in general to plants having an increasedresistance to imidazolinone herbicides. More specifically, the presentinvention relates to wheat plants obtained by mutagenesis andcross-breeding and transformation that have an increased resistance toimidazolinone herbicides.

BACKGROUND OF THE INVENTION

Acetohydroxyacid synthase (AHAS; EC 4.1.3.18) is the first enzyme thatcatalyzes the biochemical synthesis of the branched chain amino acidsvaline, leucine and isoleucine (Singh B. K., 1999 Biosynthesis ofvaline, leucine and isoleucine in: Singh B. K. (Ed) Plant amino acids.Marcel Dekker Inc. New York, N.Y. Pg 227-247). AHAS is the site ofaction of four structurally diverse herbicide families including thesulfonylureas (LaRossa RA and Falco S C, 1984 Trends Biotechnol2:158-161), the imidazolinones (Shaner et al., 1984 Plant Physiol76:545-546), the triazolopyrimidines (Subramanian and Gerwick, 1989Inhibition of acetolactate synthase by triazolopyrimidines in (ed)Whitaker J R, Sonnet P E Biocatalysis in agricultural biotechnology. ACSSymposium Series, American Chemical Society. Washington, D.C. Pg277-288), and the pyrimidyloxybenzoates (Subramanian et al., 1990 PlantPhysiol 94: 239-244). Imidazolinone and sulfonylurea herbicides arewidely used in modern agriculture due to their effectiveness at very lowapplication rates and relative non-toxicity in animals. By inhibitingAHAS activity, these families of herbicides prevent further growth anddevelopment of susceptible plants including many weed species. Severalexamples of commercially available imidazolinone herbicides are PURSUIT®(imazethapyr), SCEPTER® (imazaquin) and ARSENAL® (imazapyr). Examples ofsulfonylurea herbicides are chlorsulfuron, metsulfuron methyl,sulfometuron methyl, chlorimuron ethyl, thifensulfuron methyl,tribenuron methyl, bensulfuron methyl, nicosulfuron, ethametsulfuronmethyl, rimsulfuron, triflusulfuron methyl, triasulfuron, primisulfuronmethyl, cinosulfuron, amidosulfuron, fluzasulfuron, imazosulfuron,pyrazosulfuron ethyl and halosulfuron.

Due to their high effectiveness and low-toxicity, imidazolinoneherbicides are favored for application by spraying over the top of awide area of vegetation. The ability to spray an herbicide over the topof a wide range of vegetation decreases the costs associated withplantation establishment and maintenance and decreases the need for sitepreparation prior to use of such chemicals. Spraying over the top of adesired tolerant species also results in the ability to achieve maximumyield potential of the desired species due to the absence of competitivespecies. However, the ability to use such spray-over techniques isdependent upon the presence of imidazolinone resistant species of thedesired vegetation in the spray over area.

Among the major agricultural crops, some leguminous species such assoybean are naturally resistant to imidazolinone herbicides due to theirability to rapidly metabolize the herbicide compounds (Shaner andRobinson, 1985 Weed Sci. 33:469-471). Other crops such as corn (Newhouseet al., 1992 Plant Physiol. 100:882-886) and rice (Barrette et al., 1989Crop Safeners for Herbicides, Academic Press New York, pp. 195-220) aresomewhat susceptible to imidazolinone herbicides. The differentialsensitivity to the imidazolinone herbicides is dependent on the chemicalnature of the particular herbicide and differential metabolism of thecompound from a toxic to a non-toxic form in each plant (Shaner et al.,1984 Plant Physiol. 76:545-546; Brown et al., 1987 Pestic. Biochm.Physiol. 27:24-29). Other plant physiological differences such asabsorption and translocation also play an important role in sensitivity(Shaner and Robinson, 1985 Weed Sci. 33:469-471).

Crop cultivars resistant to imidazolinones, sulfonylureas andtriazolopyrimidines have been successfully produced using seed,microspore, pollen, and callus mutagenesis in Zea mays, Arabidopsisthaliana, Brassica napus, Glycine max, and Nicotiana tabacum (Sebastianet al., 1989 Crop Sci. 29:1403-1408; Swanson et al., 1989 Theor. Appl.Genet. 78:525-530; Newhouse et al., 1991 Theor. Appl. Genet. 83:65-70;Sathasivan et al., 1991 Plant Physiol. 97:1044-1050; Mourand et al.,1993 J. Heredity 84: 91-96). In all cases, a single, partially dominantnuclear gene conferred resistance. Four imidazolinone resistant wheatplants were also previously isolated following seed mutagenesis ofTriticum aestivum L. cv Fidel (Newhouse et al., 1992 Plant Physiol.100:882-886). Inheritance studies confirmed that a single, partiallydominant gene conferred resistance. Based on allelic studies, theauthors concluded that the mutations in the four identified lines werelocated at the same locus. One of the Fidel cultivar resistance geneswas designated FS-4 (Newhouse et al., 1992 Plant Physiol. 100:882-886).

Computer-based modeling of the three dimensional conformation of theAHAS-inhibitor complex predicts several amino acids in the proposedinhibitor binding pocket as sites where induced mutations would likelyconfer selective resistance to imidazolinones (Ott et al., 1996 J. Mol.Biol. 263:359-368) Wheat plants produced with some of these rationallydesigned mutations in the proposed binding sites of the AHAS enzyme havein fact exhibited specific resistance to a single class of herbicides(Ott et al., 1996 J. Mol. Biol. 263:359-368).

Plant resistance to imidazolinone herbicides has also been reported in anumber of patents. U.S. Pat. Nos. 4,761,373, 5,331,107, 5,304,732,6,211,438, 6,211,439 and 6,222,100 generally describe the use of analtered AHAS gene to elicit herbicide resistance in plants, andspecifically discloses certain imidazolinone resistant corn lines. U.S.Pat. No. 5,013,659 discloses plants exhibiting herbicide resistancepossessing mutations in at least one amino acid in one or more conservedregions. The mutations described therein encode either cross-resistancefor imidazolinones and sulfonylureas or sulfonylurea-specificresistance, but imidazolinone-specific resistance is not described.Additionally, U.S. Pat. No. 5,731,180 and U.S. Pat. No. 5,767,361discuss an isolated gene having a single amino acid substitution in awild-type monocot AHAS amino acid sequence that results inimidazolinone-specific resistance.

To date, the prior art has not described imidazolinone resistant wheatplants containing more than one altered AHAS gene. Nor has the prior artdescribed imidazolinone resistant wheat plants containing mutations ongenomes other than the genome from which the FS-4 gene is derived.Therefore, what is needed in the art is the identification ofimidazolinone resistance genes from additional genomes. What are alsoneeded in the art are wheat plants having increased resistance toherbicides such as imidazolinone and containing more than one alteredAHAS gene. Also needed are methods for controlling weed growth in thevicinity of such wheat plants. These compositions and methods wouldallow for the use of spray over techniques when applying herbicides toareas containing wheat plants.

SUMMARY OF THE INVENTION

The present invention provides wheat plants comprising IMI nucleicacids, wherein the wheat plant has increased resistance to animidazolinone herbicide as compared to a wild-type variety of the plant.The wheat plants can contain one, two, three or more IMI nucleic acids.In one embodiment, the wheat plant comprises multiple IMI nucleic acidslocated on different genomes. Preferably, the IMI nucleic acids encodeproteins comprising a mutation in a conserved amino acid sequenceselected from the group consisting of a Domain A, a Domain B, a DomainC, a Domain D and a Domain E. More preferably, the mutation is in aconserved Domain E or a conserved Domain C. Also provided are plantparts and plant seeds derived from the wheat plants described herein. Inanother embodiment, the wheat plant comprises an IMI nucleic acid thatis not an Imi1 nucleic acid. The IMI nucleic acid can be an Imi2 or Imi3nucleic acid, for example.

The IMI nucleic acids of the present invention can comprise a nucleotidesequence selected from the group consisting of: a polynucleotide of SEQID NO:1; a polynucleotide of SEQ ID NO:3; a polynucleotide sequenceencoding a polypeptide of SEQ ID NO:2; a polynucleotide sequenceencoding a polypeptide of SEQ ID NO:4, a polynucleotide comprising atleast 60 consecutive nucleotides of any of the aforementionedpolynucleotides; and a polynucleotide complementary to any of theaforementioned polynucleotides.

The plants of the present invention can be transgenic or non-transgenic.Examples of non-transgenic wheat plants having increased resistance toimidazolinone herbicides include a wheat plant having an ATCC PatentDeposit Designation Number PTA-3953 or PTA-3955; or a mutant,recombinant, or genetically engineered derivative of the plant with ATCCPatent Deposit Designation Number PTA-3953 or PTA-3955; or of anyprogeny of the plant with ATCC Patent Deposit Designation NumberPTA-3953 or PTA-3955; or a plant that is a progeny of any of theseplants.

In addition to the compositions of the present invention, severalmethods are provided. Described herein are methods of modifying aplant's tolerance to an imidazolinone herbicide comprising modifying theexpression of an IMI nucleic acid in the plant. Also described aremethods of producing a transgenic plant having increased tolerance to animidazolinone herbicide comprising, transforming a plant cell with anexpression vector comprising one or more IMI nucleic acids andgenerating the plant from the plant cell. The invention further includesa method of controlling weeds within the vicinity of a wheat plant,comprising applying an imidazolinone herbicide to the weeds and to thewheat plant, wherein the wheat plant has increased resistance to theimidazolinone herbicide as compared to a wild type variety of the wheatplant and wherein the plant comprises one or more IMI nucleic acids. Insome preferred embodiments of these methods, the plants comprisemultiple IMI nucleic acids that are located on different wheat genomes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table showing the results of single plant evaluation ofimazamox resistance in parental and F₁ populations resulting fromreciprocal crosses between resistant lines and CDC Teal. The numberspresented represent the number of plants scored into each phenotypicclass. Parental lines are indicated in bold. The number of parentallines scored include those scored with the F₂ populations.

FIG. 2 is a table showing the reaction to imazamox in F₂ and BCF₁populations resulting from crosses between resistant lines and CDC Tealand Chi-square tests of single locus and two locus models (15A×Teal) forcontrol of resistance. The symbols used in FIG. 2 indicate thefollowing: a—Chi-square P value (1 df) represents the probability thatdeviations from the tested ratio are due to chance alone. Chi-square Pvalues greater than 0.05 indicate that observed values were notsignificantly different from expected values; b Chi-square P valuerepresenting the probability that deviations between F₂ populationsresulting from reciprocal crosses between CDC Teal and resistant linesare due to chance alone. Chi-square values greater than 0.05 indicatethat reciprocal F₂ populations were homogeneous, and data from the tworeciprocal populations was pooled; c—CDC Teal was used as the recurrentparent; d—Ratios tested were based on the results of the F₂ generation;and e—Chi-square P value (1 df) for BCF₁ ratio.

FIG. 3 is a table showing the results of an evaluation of resistance toimazamox in F_(2:3) families resulting from crosses between resistantlines and CDC Teal and Chi-square tests of single-locus and two locusmodels (15A×Teal) for control of resistance. The symbols used in FIG. 3indicate the following: a—Family segregation ratios tested were based onthe results of the F₂ and BCF₁ populations; b—Chi-square P value (2 df)representing the probability that deviations from the tested ratio aredue to chance alone. Chi-square P values greater than 0.05 indicate thatobserved values were not significantly different from expected values.

FIG. 4 is a table showing the results of a single plant evaluation ofimazamox resistance in F₂ populations resulting from inter-crossesbetween resistant lines. Chi-square ratios tested were based on theresults of the F₂ and F_(2:3) family results obtained from crossesbetween resistant lines and CDC Teal. The 15:1 ratio tested is for a twolocus model and the 63:1 ratio tested is for a three locus model. The“a” symbol used in FIG. 4 indicates the following: Chi-square P value (1df) representing the probability that deviations from the tested ratioare due to chance alone. Chi-square P values greater than 0.05 indicatethat observed values were not significantly different from expectedvalues.

FIG. 5 is a table showing the results of an evaluation of imazamoxresistance in F_(2:3) families resulting from segregating inter-crossesbetween resistant lines. The symbols used in FIG. 5 indicate thefollowing: a—Family segregation ratios tested were based on the resultsof the F₂ populations examined; b—Chi-square P value (2 df) representingthe probability that deviations from the tested ratio are due to chancealone. Chi-square P values greater than 0.05 indicate that observedvalues were not significantly different from expected values.

FIG. 6 is a table comparing the percent uninhibited in vitro AHASactivity in four wheat lines in the presence of increasingconcentrations of the imidazolinone herbicide imazamox. Teal is a wildtype line with no tolerance to imidazolinone herbicides while BW755contains the FS4 mutant gene.

FIG. 7 is a table comparing injury sustained by three wheat genotypeswhen treated with either a 10× or 30× rate of imazamox. The 1× rate is20 g/ha. BW755 contains the FS4 mutant gene. 15A/11A is a bulk of selfedprogeny from the cross of Teal11A and Teal15A. The population was notyet homozygous at all three non-allelic loci.

FIG. 8 shows a DNA sequence alignment of partial Als1 and Imi1 wheatgenes amplified from genomic DNA: CDC Teal (row 2; SEQ ID NO:15 and SEQID NO:16), BW755 (row 3; SEQ ID NO:17 and SEQ ID NO:18), TealIMI 10A(row 4; SEQ ID NO:19 and SEQ ID NO:20), TealIMI 11A (row 5; SEQ ID NO:21and SEQ ID NO:22), and TealIMI 15A (row 6; SEQ ID NO:23 and SEQ IDNO:24). Partial sequences were aligned with a complete rice ALS genesequence (row 1; SEQ ID NO:13 and SEQ ID NO:14) derived from Genbank(Accession no. ABO49822) and translated to protein sequences (presentedon top of the DNA sequences). The five highly conserved amino aciddomains known to house mutations that confer resistance to AHASinhibitors are indicated in bold. Note the guanine to adeninesubstitutions in BW755, TealIMI 10A, and TealIMI 15A result in a serineto asparagine substitution (serine-627 in rice) in the IPSGG domain(Domain E) of the Als1 gene. Accordingly, the resistance genes presentin the BW755, TealIMI 10A, and TealIMI 15A plants have been designatedas part of the Imi1 class. These Teal resistance genes are referred toherein as TealIMI 10A and TealIMI 15A.

FIG. 9 shows a DNA sequence alignment of partial Als2 and Imi2 wheatgenes amplified from genomic DNA: CDC Teal (row 2; SEQ ID NO:25 and SEQID NO:26), BW755 (row 3; SEQ ID NO:27 and SEQ ID NO:28), TealIMI 10A(row 4; SEQ ID NO:29 and SEQ ID NO:30), TealIMI 11A (row 5; SEQ ID NO:31and SEQ ID NO:32) and TealIMI 15A (row 6; SEQ ID NO:33 and SEQ IDNO:34). Partial AHAS sequences were aligned with a complete rice AHASsequence (row 1; SEQ ID NO:13 and SEQ ID NO:14) derived from GenBank(Accession no. AB049822) and translated into protein sequences(presented above the DNA sequences). The five highly conserved domainsknown to house mutations that confer resistance to AHAS inhibitors areindicated in bold. Note the guanine to adenine substitution in TealIMI11A, resulting in a serine to asparagine substitution (serine-627 inrice) in the IPSGG domain of the Als2 gene. Accordingly, the resistancegene present in TealIMI 11A plant has been designated as part of theImi2 class of nucleic acids. This Teal resistance gene is referred toherein as TealIMI2 11A.

FIG. 10 shows the partial DNA sequence of TealIMI 15A (SEQ ID NO:1) andthe deduced amino acid sequence of the same (SEQ ID NO:2).

FIG. 11 shows the partial DNA sequence of TealIMI2 11A (SEQ ID NO:3) andthe deduced amino acid sequence of the same (SEQ ID NO:4).

FIG. 12 shows the wild type nucleic acid sequence of the Teal ALS1 ORF(SEQ ID NO:5), the Teal ALS2 ORF (SEQ ID NO:6) the Teal ALS3 ORF (SEQ IDNO:7).

FIG. 13 is a schematic representation of the conserved amino acidsequences in the AHAS genes implicated in resistance to various AHASinhibitors. The specific amino acid site responsible for resistance isindicated by an underline. (Modified from Devine, M. D. and Eberlein, C.V., 1997 Physiological, biochemical and molecular aspects of herbicideresistance based on altered target sites in Herbicide Activity Toxicity,Biochemistry, and Molecular Biology, IOS Press Amsterdam, p. 159-185).

DETAILED DESCRIPTION

The present invention is directed to wheat plants, wheat plant parts andwheat plant cells having increased resistance to imidazolinoneherbicides. The present invention also includes seeds produced by thewheat plants described herein and methods for controlling weeds in thevicinity of the wheat plants described herein. It is to be understoodthat as used in the specification and in the claims, “a” or “an” canmean one or more, depending upon the context in which it is used. Thus,for example, reference to “a cell” can mean that at least one cell canbe utilized.

As used herein, the term “wheat plant” refers to a plant that is amember of the Triticum genus. The wheat plants of the present inventioncan be members of a Triticum genus including, but not limited to, T.aestivum, T. turgidum, T. timopheevii, T. monococcum, T. zhukovskyi andT. urartu and hybrids thereof. Examples of T. aestivum subspeciesincluded within the present invention are aestivum (common wheat),compactum (club wheat), macha (macha wheat), vavilovi (vavilovi wheat),spelta and sphaecrococcum (shot wheat). Examples of T. turgidumsubspecies included within the present invention are turgidum,carthlicum, dicoccon, durum, paleocolchicum, polonicum, turanicum anddicoccoides. Examples of T. monococcum subspecies included within thepresent invention are monococcum (einkorn) and aegilopoides. In oneembodiment of the present invention, the wheat plant is a member of theTriticum aestivum species, and more particularly, the CDC Teal cultivar.

The term “wheat plant” is intended to encompass wheat plants at anystage of maturity or development as well as any tissues or organs (plantparts) taken or derived from any such plant unless otherwise clearlyindicated by context. Plant parts include, but are not limited to,stems, roots, flowers, ovules, stamens, leaves, embryos, meristematicregions, callus tissue, anther cultures, gametophytes, sporophytes,pollen, microspores, protoplasts and the like. The present inventionalso includes seeds produced by the wheat plants of the presentinvention. In one embodiment, the seeds are true breeding for anincreased resistance to an imidazolinone herbicide as compared to a wildtype variety of the wheat plant seed.

The present invention describes a wheat plant comprising one or more IMInucleic acids, wherein the wheat plant has increased resistance to animidazolinone herbicide as compared to a wild-type variety of the plant.As used herein, the term “IMI nucleic acid” refers to a nucleic acidthat is mutated from an AHAS nucleic acid in a wild type wheat plantthat confers increased imidazolinone resistance to a plant in which itis transcribed. Wild type Teal AHAS nucleic acids are shown in SEQ IDNO:5 (Teal ALS1 ORF), SEQ ID NO:6 (Teal ALS2 ORF) and SEQ ID NO:7 (TealALS3 ORF). In one embodiment, the wheat plant comprises multiple IMInucleic acids. As used when describing the IMI nucleic acids, the term“multiple” refers to IMI nucleic acids that have different nucleotidesequences and does not refer to a mere increase in number of the sameIMI nucleic acid. For example, the IMI nucleic acids can be differentdue to the fact that they are derived from or located on different wheatgenomes.

It is possible for the wheat plants of the present invention to havemultiple IMI nucleic acids from different genomes since these plants cancontain more than one genome. For example, a Triticum aestivum wheatplant contains three genomes sometimes referred to as the A, B and Dgenomes. Because AHAS is a required metabolic enzyme, it is assumed thateach genome has at least one gene coding for the AHAS enzyme, commonlyseen with other metabolic enzymes in hexaploid wheat that have beenmapped. The AHAS nucleic acid on each genome can, and usually does,differ in its nucleotide sequence from an AHAS nucleic acid on anothergenome. One of skill in the art can determine the genome of origin ofeach AHAS nucleic acid through genetic crossing and/or either sequencingmethods or exonuclease digestion methods known to those of skill in theart and as also described in Example 2 below. For the purposes of thisinvention, IMI nucleic acids derived from one of the A, B or D genomesare distinguished and designated as Imi1, Imi2 or Imi3 nucleic acids.

It is not stated herein that any particular Imi nucleic acid classcorrelates with any particular A, B or D genome. For example, it is notstated herein that the Imi1 nucleic acids correlate to A genome nucleicacids, that Imi2 nucleic acids correlate to B genome nucleic acids, etc.The Imi1, Imi2 and Imi3 designations merely indicate that the IMInucleic acids within each such class do not segregate independently,whereas two IMI nucleic acids from different classes do segregateindependently and may therefore be derived from different wheat genomes.The Imi1 class of nucleic acids includes the FS-4 gene as described byNewhouse et al. (1992 Plant Physiol. 100:882-886) and the TealIMI 15Agene described in more detail below. The Imi2 class of nucleic acidsincludes the TealIMI2 11A gene described below. Each Imi class caninclude members from different wheat species. Therefore, each imi classincludes IMI nucleic acids that differ in their nucleotide sequence butthat are nevertheless designated as originating from, or being locatedon, the same wheat genome using inheritance studies as described in theExamples below and known to those of ordinary skill in the art.

Accordingly, the present invention includes a wheat plant comprising oneor more IMI nucleic acids, wherein the wheat plant has increasedresistance to an imidazolinone herbicide as compared to a wild-typevariety of the plant and wherein the one or more IMI nucleic acids areselected from a group consisting of an Imi1, Imi2 and Imi3 nucleic acid.In one embodiment, the plant comprises an Imi1 nucleic and an Imi3nucleic acid. In a preferred embodiment, the Imi1 nucleic acid comprisesthe polynucleotide sequence shown in SEQ ID NO:1. In another embodiment,the plant comprises an Imi2 nucleic acid. In a preferred embodiment, theImi2 nucleic acid comprises the polynucleotide sequence shown in SEQ IDNO:3.

As used herein with regard to nucleic acids, the term “from” refers to anucleic acid “located on” or “derived from” a particular genome. Theterm “located on” refers to a nucleic acid contained within thatparticular genome. As also used herein with regard to a genome, the term“derived from” refers to a nucleic acid that has been removed orisolated from that genome. The term “isolated” is defined in more detailbelow.

In another embodiment, the wheat plant comprises an IMI nucleic acid,wherein the nucleic acid is a non-Imi1 nucleic acid. The term“non-Imi1”, refers to an IMI nucleic acid that is not a member of theImi1 class as described above. Examples of nucleic acids from the Imi1class are shown in rows 3, 4 and 5 of FIG. 8. One example of non-Imi1nucleic acid is shown in row 5 of FIG. 8. Accordingly, in a preferredembodiment, the wheat plant comprises an IMI nucleic acid comprising thepolynucleotide sequence encoding the polypeptide of SEQ ID NO:4. Thepolynucleotide sequence can comprise the sequence shown in SEQ ID NO:3.

The present invention includes wheat plants comprising one, two, threeor more IMI nucleic acids, wherein the wheat plant has increasedresistance to an imidazolinone herbicide as compared to a wild-typevariety of the plant. The IMI nucleic acids can comprise a nucleotidesequence selected from the group consisting of a polynucleotide of SEQID NO:1; a polynucleotide of SEQ ID NO:3; a polynucleotide sequenceencoding a polypeptide of SEQ ID NO:2; a polynucleotide sequenceencoding a polypeptide of SEQ ID NO:4, a polynucleotide comprising atleast 60 consecutive nucleotides of any of the aforementionedpolynucleotides; and a polynucleotide complementary to any of theaforementioned polynucleotides.

The imidazolinone herbicide can be selected from, but is not limited to,PURSUIT® (imazethapyr), CADRE® (imazapic), RAPTOR® (imazamox), SCEPTER®(imazaquin), ASSERT® (imazethabenz), ARSENAL® (imazapyr), a derivativeof any of the aforementioned herbicides, or a mixture of two or more ofthe aforementioned herbicides, for example, imazapyr/imazamox(ODYSSEY®). More specifically, the imidazolinone herbicide can beselected from, but is not limited to,2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-nicotinic acid,2-(4-isopropyl)-4-methyl-5-oxo-2-imidazolin-2-yl)-3-quinolinecarboxylicacid, 5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-nicotinicacid,2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-(methoxymethyl)-nicotinicacid, 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-methylnicotinicacid, and a mixture of methyl6-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-m-toluate and methyl2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-p-toluate. The use of5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-nicotinic acidand2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-(methoxymethyl)-nicotinicacid is preferred. The use of2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-(methoxymethyl)-nicotinicacid is particularly preferred.

In one embodiment, the wheat plant comprises two IMI nucleic acids,wherein the nucleic acids are derived from or located on different wheatgenomes. Preferably, one of the two nucleic acids is an Imi1 nucleicacid, and more preferably comprises the polynucleotide sequence of SEQID NO: 1. In another embodiment, the wheat plant comprises one IMInucleic acid, wherein the nucleic acid comprises the polynucleotidesequence of SEQ ID NO:1 or SEQ ID NO:3. In yet another embodiment, thewheat plant comprises three or more IMI nucleic acids wherein eachnucleic acid is from a different genome. Preferably, at least one of thethree IMI nucleic acids comprises a polynucleotide sequence selectedfrom the group consisting of SEQ ID NO:1 and SEQ ID NO:3.

In a preferred embodiment of the present invention, the one or more IMInucleic acids contained within the plant encode an amino acid sequencecomprising a mutation in a domain that is conserved among several AHASproteins. These conserved domains are referred to herein as Domain A,Domain B, Domain C, Domain D and Domain E. FIG. 13 shows the generallocation of each domain in an AHAS protein. As used herein, Domain Acontains the amino acid sequence AITGQVPRRMIGT (SEQ ID NO:8); Domain Bcontains the amino acid sequence QWED (SEQ ID NO:9); Domain C containsthe amino acid sequence VFAYPGGASMEIHQALTRS (SEQ ID NO:10); Domain Dcontains the amino acid sequence AFQETP (SEQ ID NO:11); Domain Econtains the amino acid sequence IPSGG (SEQ ID NO:12). The presentinvention also contemplates that there may be slight variations in theconserved domains, for example, in cockleberry plants, the serineresidue in Domain E is replaced by an alanine residue.

Accordingly, the present invention includes a wheat plant comprising anIMI nucleic acid that encodes an amino acid sequence having a mutationin a conserved domain selected from the group consisting of a Domain A,a Domain B, a Domain C, a Domain D and a Domain E. In one embodiment,the wheat plant comprises an IMI nucleic acid that encodes an amino acidsequence having a mutation in a Domain E. In further preferredembodiments, the mutations in the conserved domains occur at thelocations indicated by the following underlining: AITGQVPRRMIGT (SEQ IDNO:8); QWED (SEQ ID NO:9); VFAYPGGASMEIHQALTRS (SEQ ID NO:10); AFQETP(SEQ ID NO:11) and IPSGG (SEQ ID NO:12). One preferred substitution isasparagine for serine in Domain E (SEQ ID NO:12).

The wheat plants described herein can be either transgenic wheat plantsor non-transgenic wheat plants. As used herein, the term “transgenic”refers to any plant, plant cell, callus, plant tissue or plant part,that contains all or part of at least one recombinant polynucleotide. Inmany cases, all or part of the recombinant polynucleotide is stablyintegrated into a chromosome or stable extra-chromosomal element, sothat it is passed on to successive generations. For the purposes of theinvention, the term “recombinant polynucleotide” refers to apolynucleotide that has been altered, rearranged or modified by geneticengineering. Examples include any cloned polynucleotide, orpolynucleotides, that are linked or joined to heterologous sequences.The term “recombinant” does not refer to alterations of polynucleotidesthat result from naturally occurring events, such as spontaneousmutations, or from non-spontaneous mutagenesis followed by selectivebreeding. Plants containing mutations arising due to non-spontaneousmutagenesis and selective breeding are referred to herein asnon-transgenic plants and are included in the present invention. Inembodiments wherein the wheat plant is transgenic and comprises multipleIMI nucleic acids, the nucleic acids can be derived from differentgenomes or the same genome. Alternatively, in embodiments wherein thewheat plant is non-transgenic and comprises multiple IMI nucleic acids,the nucleic acids are located on different genomes.

An example of a non-transgenic wheat plant cultivar comprising one IMInucleic acid is the plant cultivar deposited with the ATCC under PatentDeposit Designation Number PTA-3953 and designated herein as the TealIMI11A wheat cultivar. The TealIMI 11A wheat cultivar contains an Imi2nucleic acid. The partial nucleotide and deduced amino acid sequencescorresponding to the TealIMI2 11A gene are shown in SEQ ID NO:3 and SEQID NO:4, respectively. The only portion of the sequences not included inSEQ ID NO:3 and SEQ ID NO:4 are those sequences encoding andcorresponding to a signal sequence that is cleaved from the matureTealIMI2 11A protein. Accordingly, SEQ ID NO:4 represents the fulldeduced sequence of the mature TealIMI2 11A protein.

An example of a wheat plant cultivar comprising two IMI nucleic acids ondifferent genomes is the plant cultivar deposited with the ATCC underPatent Deposit Designation Number PTA-3955 and designated herein as theTealIMI 15A wheat cultivar. The TealIMI 15A wheat cultivar contains Imi1and Imi3 nucleic acids. The Imi1 nucleic acid comprises a mutation thatresults in a serine to asparagine change in the IMI protein encodedthereby. The mutated AHAS genes are designated herein as TealIMI 15A andTealIMI3 15A. The partial nucleotide and deduced amino acid sequencescorresponding to the TealIMI 15A gene are shown in SEQ ID NO:1 and SEQID NO:2, respectively. The only portion of the sequences not included inSEQ ID NO:1 and SEQ ID NO:2 are those sequences encoding andcorresponding to approximately 100-150 base pairs at the 5′ end andapproximately 5 base pairs at the 3′ end of the coding region.

Separate deposits of 2500 seeds of the TealIMI 11A and TealIMI 15A wheatcultivars were made with the American Type Culture Collection, Manassas,Va. on Jan. 3, 2002. These deposits were made in accordance with theterms and provisions of the Budapest Treaty relating to the deposit ofmicroorganisms. The deposits were made for a term of at least thirtyyears and at least five years after the most recent request for thefurnishing of a sample of the deposit is received by the ATCC. Thedeposited seeds were accorded Patent Deposit Designation NumbersPTA-3953 (TealIMI 11A) and PTA-3955 (TealIMI 15A).

The present invention includes the wheat plant having a Patent DepositDesignation Number PTA-3953 or PTA-3955; a mutant, recombinant, orgenetically engineered derivative of the plant with Patent DepositDesignation Number PTA-3953 or PTA-3955; any progeny of the plant withPatent Deposit Designation Number PTA-3953 or PTA-3955; and a plant thatis the progeny of any of these plants. In a preferred embodiment, thewheat plant of the present invention additionally has the herbicideresistance characteristics of the plant with Patent Deposit DesignationNumber PTA-3953 or PTA-3955.

Also included in the present invention are hybrids of the TealIMI 11Aand TealIMI 15A wheat cultivars described herein. Example 5 demonstratesTealIMI11A/TealIMI15A hybrids having increased resistance to animidazolinone herbicide. The present invention further includes hybridsof the TealIMI 11A or TealIMI 15A wheat cultivars and another wheatcultivar. The other wheat cultivar includes, but is not limited to, T.aestivum L. cv Fidel and any wheat cultivar harboring a mutant geneFS-1, FS-2, FS-3 or FS-4. (See U.S. Pat. No. 6,339,184 and U.S. patentapplication Ser. No. 08/474,832). In a preferred embodiment, the wheatplant is a hybrid between a TealIMI 11A cultivar and a Fidel FS-4cultivar. The TealIMI 11A/FS-4 hybrids comprise an Imi1 nucleic acid andan Imi2 nucleic acid. A hybrid of TealIMI 11A and a Fidel cultivarharboring the FS-4 gene is included in the present invention and wasdeposited with the American Type Culture Collection, Manassas, Va. onJan. 3, 2002. This deposit was made in accordance with the terms andprovisions of the Budapest Treaty relating to the deposit ofmicroorganisms. The deposit was made for a term of at least thirty yearsand at least five years after the most recent request for the furnishingof a sample of the deposit is received by the ATCC. The deposited seedswere accorded Patent Deposit Designation Number PTA-3954.

The terms “cultivar” and “variety” refer to a group of plants within aspecies defined by the sharing of a common set of characteristics ortraits accepted by those skilled in the art as sufficient to distinguishone cultivar or variety from another cultivar or variety. There is noimplication in either term that all plants of any given cultivar orvariety will be genetically identical at either the whole gene ormolecular level or that any given plant will be homozygous at all loci.A cultivar or variety is considered “true breeding” for a particulartrait if, when the true-breeding cultivar or variety is self-pollinated,all of the progeny contain the trait. In the present invention, thetrait arises from a mutation in an AHAS gene of the wheat plant or seed.

It is to be understood that the wheat plant of the present invention cancomprise a wild type or non-mutated AHAS gene in addition to an IMIgene. As described in Example 4, it is contemplated that wheat cultivarTealIMI 11A contains a mutation in only one of multiple AHAS isoenzymesand that wheat cultivar TealIMI 15A contains a mutation in only two ofmultiple AHAS isoenzymes. Therefore, the present invention includes awheat plant comprising one or more IMI nucleic acids in addition to oneor more wild type or non-mutated AHAS nucleic acids.

In addition to wheat plants, the present invention encompasses isolatedIMI proteins and nucleic acids. The nucleic acids comprise apolynucleotide selected from the group consisting of a polynucleotide ofSEQ ID NO:1; a polynucleotide of SEQ ID NO:3; a polynucleotide sequenceencoding a polypeptide of SEQ ID NO:2; a polynucleotide sequenceencoding a polypeptide of SEQ ID NO:4, a polynucleotide comprising atleast 60 consecutive nucleotides of any of the aforementionedpolynucleotides; and a polynucleotide complementary to any of theaforementioned polynucleotides. In a preferred embodiment, the IMInucleic acid comprises a polynucleotide sequence encoding a polypeptideof SEQ ID NO:2 or SEQ ID NO:4. In a further preferred embodiment, theIMI nucleic acid comprises a polynucleotide sequence of SEQ ID NO:1 orSEQ ID NO:3.

The term “AHAS protein” refers to an acetohydroxyacid synthase proteinand the term “IMI protein” refers to any AHAS protein that is mutatedfrom a wild type AHAS protein and that confers increased imidazolinoneresistance to a plant, plant cell, plant part, plant seed or planttissue when it is expressed therein. In a preferred embodiment, the IMIprotein comprises a polypeptide of SEQ ID NO:2 or SEQ ID NO:4. As alsoused herein, the terms “nucleic acid” and “polynucleotide” refer to RNAor DNA that is linear or branched, single or double stranded, or ahybrid thereof. The term also encompasses RNA/DNA hybrids. These termsalso encompass untranslated sequence located at both the 3′ and 5′ endsof the coding region of the gene: at least about 1000 nucleotides ofsequence upstream from the 5′ end of the coding region and at leastabout 200 nucleotides of sequence downstream from the 3′ end of thecoding region of the gene. Less common bases, such as inosine,5-methylcytosine, 6-methyladenine, hypoxanthine and others can also beused for antisense, dsRNA and ribozyme pairing. For example,polynucleotides that contain C-5 propyne analogues of uridine andcytidine have been shown to bind RNA with high affinity and to be potentantisense inhibitors of gene expression. Other modifications, such asmodification to the phosphodiester backbone, or the 2′-hydroxy in theribose sugar group of the RNA can also be made. The antisensepolynucleotides and ribozymes can consist entirely of ribonucleotides,or can contain mixed ribonucleotides and deoxyribonucleotides. Thepolynucleotides of the invention may be produced by any means, includinggenomic preparations, cDNA preparations, in vitro synthesis, RT-PCR andin vitro or in vivo transcription.

An “isolated” nucleic acid molecule is one that is substantiallyseparated from other nucleic acid molecules, which are present in thenatural source of the nucleic acid (i.e., sequences encoding otherpolypeptides). Preferably, an “isolated” nucleic acid is free of some ofthe sequences that naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in its naturallyoccurring replicon. For example, a cloned nucleic acid is consideredisolated. In various embodiments, the isolated IMI nucleic acid moleculecan contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1kb of nucleotide sequences which naturally flank the nucleic acidmolecule in genomic DNA of the cell from which the nucleic acid isderived (e.g., a Triticum aestivum cell). A nucleic acid is alsoconsidered isolated if it has been altered by human intervention, orplaced in a locus or location that is not its natural site, or if it isintroduced into a cell by agroinfection or biolistics. Moreover, an“isolated” nucleic acid molecule, such as a cDNA molecule, can be freefrom some of the other cellular material with which it is naturallyassociated, or culture medium when produced by recombinant techniques,or chemical precursors or other chemicals when chemically synthesized.

Specifically excluded from the definition of “isolated nucleic acids”are: naturally-occurring chromosomes (such as chromosome spreads),artificial chromosome libraries, genomic libraries, and cDNA librariesthat exist either as an in vitro nucleic acid preparation or as atransfected/transformed host cell preparation, wherein the host cellsare either an in vitro heterogeneous preparation or plated as aheterogeneous population of single colonies. Also specifically excludedare the above libraries wherein a specified nucleic acid makes up lessthan 5% of the number of nucleic acid inserts in the vector molecules.Further specifically excluded are whole cell genomic DNA or whole cellRNA preparations (including whole cell preparations that aremechanically sheared or enzymatically digested). Even furtherspecifically excluded are the whole cell preparations found as either anin vitro preparation or as a heterogeneous mixture separated byelectrophoresis wherein the nucleic acid of the invention has notfurther been separated from the heterologous nucleic acids in theelectrophoresis medium (e.g., further separating by excising a singleband from a heterogeneous band population in an agarose gel or nylonblot).

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule containing a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3 ora portion thereof, can be isolated using standard molecular biologytechniques and the sequence information provided herein. For example, aT. aestivum IMI cDNA can be isolated from a T. aestivum library usingall or a portion of the sequence of SEQ ID NO:1 or SEQ ID NO:3.Moreover, a nucleic acid molecule encompassing all or a portion of SEQID NO:1 or SEQ ID NO:3 can be isolated by the polymerase chain reactionusing oligonucleotide primers designed based upon this sequence. Forexample, mRNA can be isolated from plant cells (e.g., by theguanidinium-thiocyanate extraction procedure of Chirgwin et al., 1979Biochemistry 18:5294-5299) and cDNA can be prepared using reversetranscriptase (e.g., Moloney MLV reverse transcriptase, available fromGibco/BRL, Bethesda, Md.; or AMV reverse transcriptase, available fromSeikagaku America, Inc., St. Petersburg, Fla.). Syntheticoligonucleotide primers for polymerase chain reaction amplification canbe designed based upon the nucleotide sequence shown in SEQ ID NO:1 orSEQ ID NO:3. A nucleic acid molecule of the invention can be amplifiedusing cDNA or, alternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid molecule so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to an IMI nucleotidesequence can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

The IMI nucleic acids of the present invention can comprise sequencesencoding an IMI protein (i.e., “coding regions”), as well as 5′untranslated sequences and 3′ untranslated sequences. Alternatively, thenucleic acid molecules of the present invention can comprise only thecoding regions of an IMI gene, or can contain whole genomic fragmentsisolated from genomic DNA. A coding region of these sequences isindicated as an “ORF position”. Moreover, the nucleic acid molecule ofthe invention can comprise a portion of a coding region of an IMI gene,for example, a fragment that can be used as a probe or primer. Thenucleotide sequences determined from the cloning of the IMI genes fromT. aestivum allow for the generation of probes and primers designed foruse in identifying and/or cloning IMI homologs in other cell types andorganisms, as well as IMI homologs from other wheat plants and relatedspecies. The portion of the coding region can also encode a biologicallyactive fragment of an IMI protein.

As used herein, the term “biologically active portion of” an IMI proteinis intended to include a portion, e.g., a domain/motif, of an IMIprotein that, when produced in a plant increases the plant's resistanceto an imidazolinone herbicide as compared to a wild-type variety of theplant. Methods for quantitating increased resistance to imidazolinoneherbicides are provided in the Examples provided below. Biologicallyactive portions of an IMI protein include peptides encoded bypolynucleotide sequences comprising SEQ ID NO:1 or SEQ ID NO:3 whichinclude fewer amino acids than a full length IMI protein and impartincreased resistance to an imidazolinone herbicide upon expression in aplant. Typically, biologically active portions (e.g., peptides whichare, for example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 ormore amino acids in length) comprise a domain or motif with at least oneactivity of an IMI protein. Moreover, other biologically active portionsin which other regions of the polypeptide are deleted, can be preparedby recombinant techniques and evaluated for one or more of theactivities described herein. Preferably, the biologically activeportions of an IMI protein include one or more conserved domainsselected from the group consisting of a Domain A, a Domain B, a DomainC, a Domain D and a Domain E, wherein the conserved domain contains amutation.

The invention also provides IMI chimeric or fusion polypeptides. As usedherein, an IMI “chimeric polypeptide” or “fusion polypeptide” comprisesan IMI polypeptide operatively linked to a non-IMI polypeptide. A“non-IMI polypeptide” refers to a polypeptide having an amino acidsequence that is not substantially identical to an IMI polypeptide,e.g., a polypeptide that is not an IMI isoenzyme, which peptide performsa different function than an IMI polypeptide. Within the fusionpolypeptide, the term “operatively linked” is intended to indicate thatthe IMI polypeptide and the non-IMI polypeptide are fused to each otherso that both sequences fulfill the proposed function attributed to thesequence used. The non-IMI polypeptide can be fused to the N-terminus orC-terminus of the IMI polypeptide. For example, in one embodiment, thefusion polypeptide is a GST-IMI fusion polypeptide in which the IMIsequence is fused to the C-terminus of the GST sequence. Such fusionpolypeptides can facilitate the purification of recombinant IMIpolypeptides. In another embodiment, the fusion polypeptide is an IMIpolypeptide containing a heterologous signal sequence at its N-terminus.In certain host cells (e.g., mammalian host cells), expression and/orsecretion of an IMI polypeptide can be increased through use of aheterologous signal sequence.

An isolated nucleic acid molecule encoding an IMI polypeptide havingsequence identity to a polypeptide encoded by a polynucleotide sequenceof SEQ ID NO:1 or SEQ ID NO:3 can be created by introducing one or morenucleotide substitutions, additions or deletions into a nucleotidesequence of SEQ ID NO:1 or SEQ ID NO:3 such that one or more amino acidsubstitutions, additions or deletions are introduced into the encodedpolypeptide. Mutations can be introduced into a sequence of SEQ ID NO:1or SEQ ID NO:3 by standard techniques, such as site-directed mutagenesisand PCR-mediated mutagenesis. Preferably, conservative amino acidsubstitutions are made at one or more predicted non-essential amino acidresidues.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in an IMI polypeptide ispreferably replaced with another amino acid residue from the same sidechain family. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of an IMI coding sequence, such asby saturation mutagenesis, and the resultant mutants can be screened foran IMI activity described herein to identify mutants that retain IMIactivity. Following mutagenesis of the sequence of SEQ ID NO:1 or SEQ IDNO:3, the encoded polypeptide can be expressed recombinantly and theactivity of the polypeptide can be determined by analyzing theimidazolinone resistance of a plant expressing the polypeptide asdescribed in the Examples below.

To determine the percent sequence identity of two amino acid sequences(e.g., SEQ ID NO:2 or SEQ ID NO:4 and a mutant form thereof), thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in the sequence of one polypeptide for optimal alignment withthe other polypeptide). The amino acid residues at corresponding aminoacid positions are then compared. When a position in one sequence (e.g.,SEQ ID NO:2 or SEQ ID NO:4) is occupied by the same amino acid residueas the corresponding position in the other sequence (e.g., a mutant formof SEQ ID NO:2 or SEQ ID NO:4), then the molecules are identical at thatposition. The same type of comparison can be made between two nucleicacid sequences. The percent sequence identity between the two sequencesis a function of the number of identical positions shared by thesequences (i.e., percent sequence identity=numbers of identicalpositions/total numbers of positions×100). For the purposes of theinvention, the percent sequence identity between two nucleic acid orpolypeptide sequences is determined using the Vector NTI 6.0 (PC)software package (InforMax, 7600 Wisconsin Ave., Bethesda, Md. 20814). Agap opening penalty of 15 and a gap extension penalty of 6.66 are usedfor determining the percent identity of two nucleic acids. A gap openingpenalty of 10 and a gap extension penalty of 0.1 are used fordetermining the percent identity of two polypeptides. All otherparameters are set at the default settings. It is to be understood thatfor the purposes of determining sequence identity, when comparing a DNAsequence to an RNA sequence, a thymidine nucleotide is equivalent to auracil nucleotide. Preferably, the isolated IMI polypeptides included inthe present invention are at least about 50-60%, preferably at leastabout 60-70%, and more preferably at least about 70-75%, 75-80%, 80-85%,85-90% or 90-95%, and most preferably at least about 96%, 97%, 98%, 99%or more identical to an entire amino acid sequence encoded by apolynucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3. In anotherembodiment, the isolated IMI polypeptides included in the presentinvention are at least about 50-60%, preferably at least about 60-70%,and more preferably at least about 70-75%, 75-80%, 80-85%, 85-90% or90-95%, and most preferably at least about 96%, 97%, 98%, 99% or moreidentical to an entire amino acid sequence shown in SEQ ID NO:2 or SEQID NO:4.

Additionally, optimized IMI nucleic acids can be created. Preferably, anoptimized IMI nucleic acid encodes an IMI polypeptide that modulates aplant's tolerance to imidazolinone herbicides, and more preferablyincreases a plant's tolerance to an imidazolinone herbicide upon itsover-expression in the plant. As used herein, “optimized” refers to anucleic acid that is genetically engineered to increase its expressionin a given plant or animal. To provide plant optimized IMI nucleicacids, the DNA sequence of the gene can be modified to 1) comprisecodons preferred by highly expressed plant genes; 2) comprise an A+Tcontent in nucleotide base composition to that substantially found inplants; 3) form a plant initiation sequence, 4) eliminate sequences thatcause destabilization, inappropriate polyadenylation, degradation andtermination of RNA, or that form secondary structure hairpins or RNAsplice sites. Increased expression of IMI nucleic acids in plants can beachieved by utilizing the distribution frequency of codon usage inplants in general or a particular plant. Methods for optimizing nucleicacid expression in plants can be found in EPA 0359472; EPA 0385962; PCTApplication No. WO 91/16432; U.S. Pat. No. 5,380,831; U.S. Pat. No.5,436,391; Perlack et al., 1991 Proc. Natl. Acad. Sci. USA 88:3324-3328;and Murray et al., 1989 Nucleic Acids Res. 17:477-498.

As used herein, “frequency of preferred codon usage” refers to thepreference exhibited by a specific host cell in usage of nucleotidecodons to specify a given amino acid. To determine the frequency ofusage of a particular codon in a gene, the number of occurrences of thatcodon in the gene is divided by the total number of occurrences of allcodons specifying the same amino acid in the gene. Similarly, thefrequency of preferred codon usage exhibited by a host cell can becalculated by averaging frequency of preferred codon usage in a largenumber of genes expressed by the host cell. It is preferable that thisanalysis be limited to genes that are highly expressed by the host cell.The percent deviation of the frequency of preferred codon usage for asynthetic gene from that employed by a host cell is calculated first bydetermining the percent deviation of the frequency of usage of a singlecodon from that of the host cell followed by obtaining the averagedeviation over all codons. As defined herein, this calculation includesunique codons (i.e., ATG and TGG). In general terms, the overall averagedeviation of the codon usage of an optimized gene from that of a hostcell is calculated using the equation 1A=n 1 Z X_(n)−Y_(n)X_(n) times100 Z where X_(n)=frequency of usage for codon n in the host cell;Y_(n)=frequency of usage for codon n in the synthetic gene, n representsan individual codon that specifies an amino acid and the total number ofcodons is Z. The overall deviation of the frequency of codon usage, A,for all amino acids should preferably be less than about 25%, and morepreferably less than about 10%.

Hence, an IMI nucleic acid can be optimized such that its distributionfrequency of codon usage deviates, preferably, no more than 25% fromthat of highly expressed plant genes and, more preferably, no more thanabout 10%. In addition, consideration is given to the percentage G+Ccontent of the degenerate third base (monocotyledons appear to favor G+Cin this position, whereas dicotyledons do not). It is also recognizedthat the XCG (where X is A, T, C, or G) nucleotide is the leastpreferred codon in dicots whereas the XTA codon is avoided in bothmonocots and dicots. Optimized IMI nucleic acids of this invention alsopreferably have CG and TA doublet avoidance indices closelyapproximating those of the chosen host plant (i.e., Triticum aestivum).More preferably these indices deviate from that of the host by no morethan about 10-15%.

In addition to the nucleic acid molecules encoding the IMI polypeptidesdescribed above, another aspect of the invention pertains to isolatednucleic acid molecules that are antisense thereto. Antisensepolynucleotides are thought to inhibit gene expression of a targetpolynucleotide by specifically binding the target polynucleotide andinterfering with transcription, splicing, transport, translation and/orstability of the target polynucleotide. Methods are described in theprior art for targeting the antisense polynucleotide to the chromosomalDNA, to a primary RNA transcript or to a processed mRNA. Preferably, thetarget regions include splice sites, translation initiation codons,translation termination codons, and other sequences within the openreading frame.

The term “antisense”, for the purposes of the invention, refers to anucleic acid comprising a polynucleotide that is sufficientlycomplementary to all or a portion of a gene, primary transcript orprocessed mRNA, so as to interfere with expression of the endogenousgene. “Complementary” polynucleotides are those that are capable of basepairing according to the standard Watson-Crick complementarity rules.Specifically, purines will base pair with pyrimidines to form acombination of guanine paired with cytosine (G:C) and adenine pairedwith either thymine (A:T) in the case of DNA, or adenine paired withuracil (A:U) in the case of RNA. It is understood that twopolynucleotides may hybridize to each other even if they are notcompletely complementary to each other, provided that each has at leastone region that is substantially complementary to the other. The term“antisense nucleic acid” includes single stranded RNA as well asdouble-stranded DNA expression cassettes that can be transcribed toproduce an antisense RNA. “Active” antisense nucleic acids are antisenseRNA molecules that are capable of selectively hybridizing with a primarytranscript or mRNA encoding a polypeptide having at least 80% sequenceidentity with the polypeptide encoded by the polynucleotide sequence ofSEQ ID NO:1 or SEQ ID NO:3.

In addition to the IMI nucleic acids and polypeptides described above,the present invention encompasses these nucleic acids and polypeptidesattached to a moiety. These moieties include, but are not limited to,detection moieties, hybridization moieties, purification moieties,delivery moieties, reaction moieties, binding moieties, and the like. Atypical group of nucleic acids having moieties attached are probes andprimers. Probes and primers typically comprise a substantially isolatedoligonucleotide. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, preferably about 25, more preferably about 40, 50 or 75consecutive nucleotides of a sense strand of the sequence set forth inSEQ ID NO:1 or SEQ ID NO:3, an anti-sense sequence of the sequence setforth in SEQ ID NO:1 or SEQ ID NO:3, or naturally occurring mutantsthereof. Primers based on a nucleotide sequence of SEQ ID NO:1 or SEQ IDNO:3 can be used in PCR reactions to clone IMI homologs. Probes based onthe IMI nucleotide sequences can be used to detect transcripts orgenomic sequences encoding the same or homologous polypeptides. Inpreferred embodiments, the probe further comprises a label groupattached thereto, e.g. the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as a part of a genomic marker test kit for identifying cellswhich express an IMI polypeptide, such as by measuring a level of anIMI-encoding nucleic acid, in a sample of cells, e.g., detecting IMImRNA levels or determining whether a genomic IMI gene has been mutatedor deleted.

The invention further provides an isolated recombinant expression vectorcomprising an IMI nucleic acid as described above, wherein expression ofthe vector in a host cell results in increased resistance to animidazolinone herbicide as compared to a wild type variety of the hostcell. As used herein, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operably linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel, Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990) or see:Gruber and Crosby, in: Methods in Plant Molecular Biology andBiotechnology, eds. Glick and Thompson, Chapter 7, 89-108, CRC Press:Boca Raton, Fla., including the references therein. Regulatory sequencesinclude those that direct constitutive expression of a nucleotidesequence in many types of host cells and those that direct expression ofthe nucleotide sequence only in certain host cells or under certainconditions. It will be appreciated by those skilled in the art that thedesign of the expression vector can depend on such factors as the choiceof the host cell to be transformed, the level of expression ofpolypeptide desired, etc. The expression vectors of the invention can beintroduced into host cells to thereby produce polypeptides or peptides,including fusion polypeptides or peptides, encoded by nucleic acids asdescribed herein (e.g., IMI polypeptides, fusion polypeptides, etc.).

In a preferred embodiment of the present invention, the IMI polypeptidesare expressed in plants and plants cells such as unicellular plant cells(such as algae) (see Falciatore et al., 1999 Marine Biotechnology1(3):239-251 and references therein) and plant cells from higher plants(e.g., the spermatophytes, such as crop plants). An IMI polynucleotidemay be “introduced” into a plant cell by any means, includingtransfection, transformation or transduction, electroporation, particlebombardment, biolistics, agroinfection and the like. One transformationmethod known to those of skill in the art is the dipping of a floweringplant into an Agrobacteria solution, wherein the Agrobacteria containsthe IMI nucleic acid, followed by breeding of the transformed gametes.

Other suitable methods for transforming or transfecting host cellsincluding plant cells can be found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2^(nd), ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989) and other laboratory manuals such as Methods in MolecularBiology, 1995, Vol. 44, Agrobacterium protocols, ed: Gartland and Davey,Humana Press, Totowa, N.J. As increased resistance to imidazolinoneherbicides is a general trait wished to be inherited into a wide varietyof plants like maize, wheat, rye, oat, triticale, rice, barley, soybean,peanut, cotton, rapeseed and canola, manihot, pepper, sunflower andtagetes, solanaceous plants like potato, tobacco, eggplant, and tomato,Vicia species, pea, alfalfa, bushy plants (coffee, cacao, tea), Salixspecies, trees (oil palm, coconut), perennial grasses and forage crops,these crop plants are also preferred target plants for a geneticengineering as one further embodiment of the present invention. Foragecrops include, but are not limited to, Wheatgrass, Canarygrass,Bromegrass, Wildrye Grass, Bluegrass, Orchardgrass, Alfalfa, Salfoin,Birdsfoot Trefoil, Alsike Clover, Red Clover and Sweet Clover.

In one embodiment of the present invention, transfection of an IMIpolynucleotide into a plant is achieved by Agrobacterium mediated genetransfer. Agrobacterium mediated plant transformation can be performedusing for example the GV3101 (pMP90) (Koncz and Schell, 1986 Mol. Gen.Genet. 204:383-396) or LBA4404 (Clontech) Agrobacterium tumefaciensstrain. Transformation can be performed by standard transformation andregeneration techniques (Deblaere et al., 1994 Nucl. Acids. Res.13:4777-4788; Gelvin, Stanton B. and Schilperoort, Robert A, PlantMolecular Biology Manual, 2^(nd) Ed. —Dordrecht: Kluwer Academic Publ.,1995. —in Sect., Ringbuc Zentrale Signatur: BT11-P ISBN 0-7923-27314;Glick, Bernard R. and Thompson, John E., Methods in Plant MolecularBiology and Biotechnology, Boca Raton: CRC Press, 1993 360 S., ISBN0-8493-5164-2). For example, rapeseed can be transformed via cotyledonor hypocotyl transformation (Moloney et al., 1989 Plant cell Report8:238-242; De Block et al., 1989 Plant Physiol. 91:694-701). Use ofantibiotica for Agrobacterium and plant selection depends on the binaryvector and the Agrobacterium strain used for transformation. Rapeseedselection is normally performed using kanamycin as selectable plantmarker. Agrobacterium mediated gene transfer to flax can be performedusing, for example, a technique described by Mlynarova et al., 1994Plant Cell Report 13:282-285. Additionally, transformation of soybeancan be performed using, for example, a technique described in EuropeanPatent No. 0424 047, U.S. Pat. No. 5,322,783, European Patent No. 0397687, U.S. Pat. No. 5,376,543 or U.S. Pat. No. 5,169,770. Transformationof maize can be achieved by particle bombardment, polyethylene glycolmediated DNA uptake or via the silicon carbide fiber technique. (See,for example, Freeling and Walbot “The maize handbook” Springer Verlag:New York (1993) ISBN 3-540-97826-7). A specific example of maizetransformation is found in U.S. Pat. No. 5,990,387 and a specificexample of wheat transformation can be found in PCT Application No. WO93/07256.

According to the present invention, the introduced IMI polynucleotidemay be maintained in the plant cell stably if it is incorporated into anon-chromosomal autonomous replicon or integrated into the plantchromosomes. Alternatively, the introduced IMI polynucleotide may bepresent on an extra-chromosomal non-replicating vector and betransiently expressed or transiently active. In one embodiment, ahomologous recombinant microorganism can be created wherein the IMIpolynucleotide is integrated into a chromosome, a vector is preparedwhich contains at least a portion of an AHAS gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the endogenous AHAS gene and to create an IMIgene. To create a point mutation via homologous recombination, DNA-RNAhybrids can be used in a technique known as chimeraplasty (Cole-Strausset al., 1999 Nucleic Acids Research 27(5):1323-1330 and Kmiec, 1999 Genetherapy American Scientist 87(3):240-247). Other homologousrecombination procedures in Triticum species are also well known in theart and are contemplated for use herein.

In the homologous recombination vector, the IMI gene can be flanked atits 5′ and 3′ ends by an additional nucleic acid molecule of the AHASgene to allow for homologous recombination to occur between theexogenous IMI gene carried by the vector and an endogenous AHAS gene, ina microorganism or plant. The additional flanking AHAS nucleic acidmolecule is of sufficient length for successful homologous recombinationwith the endogenous gene. Typically, several hundreds of base pairs upto kilobases of flanking DNA (both at the 5′ and 3′ ends) are includedin the vector (see e.g., Thomas, K. R., and Capecchi, M. R., 1987 Cell51:503 for a description of homologous recombination vectors or Streppet al., 1998 PNAS, 95(8):4368-4373 for cDNA based recombination inPhyscomitrella patens). However, since the IMI gene normally differsfrom the AHAS gene at very few amino acids, a flanking sequence is notalways necessary. The homologous recombination vector is introduced intoa microorganism or plant cell (e.g., via polyethylene glycol mediatedDNA), and cells in which the introduced IMI gene has homologouslyrecombined with the endogenous AHAS gene are selected using art-knowntechniques.

In another embodiment, recombinant microorganisms can be produced thatcontain selected systems that allow for regulated expression of theintroduced gene. For example, inclusion of an IMI gene on a vectorplacing it under control of the lac operon permits expression of the IMIgene only in the presence of IPTG. Such regulatory systems are wellknown in the art.

Whether present in an extra-chromosomal non-replicating vector or avector that is integrated into a chromosome, the IMI polynucleotidepreferably resides in a plant expression cassette. A plant expressioncassette preferably contains regulatory sequences capable of drivinggene expression in plant cells that are operably linked so that eachsequence can fulfill its function, for example, termination oftranscription by polyadenylation signals. Preferred polyadenylationsignals are those originating from Agrobacterium tumefaciens t-DNA suchas the gene 3 known as octopine synthase of the Ti-plasmid pTiACH5(Gielen et al., 1984 EMBO J. 3:835) or functional equivalents thereof,but also all other terminators functionally active in plants aresuitable. As plant gene expression is very often not limited ontranscriptional levels, a plant expression cassette preferably containsother operably linked sequences like translational enhancers such as theoverdrive-sequence containing the 5′-untranslated leader sequence fromtobacco mosaic virus enhancing the polypeptide per RNA ratio (Gallie etal., 1987 Nucl. Acids Research 15:8693-8711). Examples of plantexpression vectors include those detailed in: Becker, D. et al., 1992New plant binary vectors with selectable markers located proximal to theleft border, Plant Mol. Biol. 20:1195-1197; Bevan, M. W., 1984 BinaryAgrobacterium vectors for plant transformation, Nucl. Acid. Res.12:8711-8721; and Vectors for Gene Transfer in Higher Plants; in:Transgenic Plants, Vol. 1, Engineering and Utilization, eds.: Kung andR. Wu, Academic Press, 1993, S. 15-38.

Plant gene expression should be operably linked to an appropriatepromoter conferring gene expression in a timely, cell or tissue specificmanner. Promoters useful in the expression cassettes of the inventioninclude any promoter that is capable of initiating transcription in aplant cell. Such promoters include, but are not limited to those thatcan be obtained from plants, plant viruses and bacteria that containgenes that are expressed in plants, such as Agrobacterium and Rhizobium.

The promoter may be constitutive, inducible, developmentalstage-preferred, cell type-preferred, tissue-preferred ororgan-preferred. Constitutive promoters are active under mostconditions. Examples of constitutive promoters include the CaMV 19S and35 S promoters (Odell et al. 1985 Nature 313:810-812), the sX CaMV 35Spromoter (Kay et al. 1987 Science 236:1299-1302) the Sep1 promoter, therice actin promoter (McElroy et al. 1990 Plant Cell 2:163-171), theArabidopsis actin promoter, the ubiquitan promoter (Christensen et al.1989 Plant Molec Biol. 18:675-689); pEmu (Last et al. 1991 Theor ApplGenet. 81:581-588), the figwort mosaic virus 35S promoter, the Smaspromoter (Velten et al. 1984 EMBO J. 3:2723-2730), the GRP1-8 promoter,the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439),promoters from the T-DNA of Agrobacterium, such as mannopine synthase,nopaline synthase, and octopine synthase, the small subunit of ribulosebiphosphate carboxylase (ssuRUBISCO) promoter, and the like.

Inducible promoters are active under certain environmental conditions,such as the presence or absence of a nutrient or metabolite, heat orcold, light, pathogen attack, anaerobic conditions, and the like. Forexample, the hsp80 promoter from Brassica is induced by heat shock, thePPDK promoter is induced by light, the PR-1 promoter from tobacco,Arabidopsis and maize are inducible by infection with a pathogen, andthe Adh1 promoter is induced by hypoxia and cold stress. Plant geneexpression can also be facilitated via an inducible promoter (for reviewsee Gatz, 1997 Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89-108).Chemically inducible promoters are especially suitable if geneexpression is wanted to occur in a time specific manner. Examples ofsuch promoters are a salicylic acid inducible promoter (PCT ApplicationNo. WO 95/19443), a tetracycline inducible promoter (Gatz et al., 1992Plant J. 2:397-404) and an ethanol inducible promoter (PCT ApplicationNo. WO 93/21334).

Developmental stage-preferred promoters are preferentially expressed atcertain stages of development. Tissue and organ preferred promotersinclude those that are preferentially expressed in certain tissues ororgans, such as leaves, roots, seeds, or xylem. Examples of tissuepreferred and organ preferred promoters include, but are not limited tofruit-preferred, ovule-preferred, male tissue-preferred, seed-preferred,integument-preferred, tuber-preferred, stalk-preferred,pericarp-preferred, and leaf-preferred, stigma-preferred,pollen-preferred, anther-preferred, a petal-preferred, sepal-preferred,pedicel-preferred, silique-preferred, stem-preferred, root-preferredpromoters and the like. Seed preferred promoters are preferentiallyexpressed during seed development and/or germination. For example, seedpreferred promoters can be embryo-preferred, endosperm preferred andseed coat-preferred. See Thompson et al. 1989 BioEssays 10:108. Examplesof seed preferred promoters include, but are not limited to cellulosesynthase (ce1A), Cim1, gamma-zein, globulin-1, maize 19 kD zein (cZ19B1)and the like.

Other suitable tissue-preferred or organ-preferred promoters include thenapin-gene promoter from rapeseed (U.S. Pat. No. 5,608,152), theUSP-promoter from Vicia faba (Baeumlein et al., 1991 Mol Gen Genet.225(3):459-67), the oleosin-promoter from Arabidopsis (PCT ApplicationNo. WO 98/45461), the phaseolin-promoter from Phaseolus vulgaris (U.S.Pat. No. 5,504,200), the Bce4-promoter from Brassica (PCT ApplicationNo. WO 91/13980) or the legumin B4 promoter (LeB4; Baeumlein et al.,1992 Plant Journal, 2(2):233-9) as well as promoters conferring seedspecific expression in monocot plants like maize, barley, wheat, rye,rice, etc. Suitable promoters to note are the lpt2 or lpt1-gene promoterfrom barley (PCT Application No. WO 95/15389 and PCT Application No. WO95/23230) or those described in PCT Application No. WO 99/16890(promoters from the barley hordein-gene, rice glutelin gene, rice oryzingene, rice prolamin gene, wheat gliadin gene, wheat glutelin gene, oatglutelin gene, Sorghum kasirin-gene and rye secalin gene).

Other promoters useful in the expression cassettes of the inventioninclude, but are not limited to, the major chlorophyll a/b bindingprotein promoter, histone promoters, the Ap3 promoter, the β-conglycinpromoter, the napin promoter, the soy bean lectin promoter, the maize 15kD zein promoter, the 22 kD zein promoter, the 27 kD zein promoter, theg-zein promoter, the waxy, shrunken 1, shrunken 2 and bronze promoters,the Zm13 promoter (U.S. Pat. No. 5,086,169), the maize polygalacturonasepromoters (PG) (U.S. Pat. Nos. 5,412,085 and 5,545,546) and the SGB6promoter (U.S. Pat. No. 5,470,359), as well as synthetic or othernatural promoters.

Additional flexibility in controlling heterologous gene expression inplants may be obtained by using DNA binding domains and responseelements from heterologous sources (i.e., DNA binding domains fromnon-plant sources). An example of such a heterologous DNA binding domainis the LexA DNA binding domain (Brent and Ptashne, Cell 43:729-736(1985)).

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but they also apply to the progeny or potentialprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein. A host cell can be any prokaryotic or eukaryotic cell. Forexample, an IMI polynucleotide can be expressed in bacterial cells suchas C. glutamicum, insect cells, fungal cells or mammalian cells (such asChinese hamster ovary cells (CHO) or COS cells), algae, ciliates, plantcells, fungi or other microorganisms like C. glutamicum. Other suitablehost cells are known to those skilled in the art.

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) an IMIpolynucleotide. Accordingly, the invention further provides methods forproducing IMI polypeptides using the host cells of the invention. In oneembodiment, the method comprises culturing the host cell of invention(into which a recombinant expression vector encoding an IMI polypeptidehas been introduced, or into which genome has been introduced a geneencoding a wild-type or IMI polypeptide) in a suitable medium until IMIpolypeptide is produced. In another embodiment, the method furthercomprises isolating IMI polypeptides from the medium or the host cell.Another aspect of the invention pertains to isolated IMI polypeptides,and biologically active portions thereof. An “isolated” or “purified”polypeptide or biologically active portion thereof is free of some ofthe cellular material when produced by recombinant DNA techniques, orchemical precursors or other chemicals when chemically synthesized. Thelanguage “substantially free of cellular material” includes preparationsof IMI polypeptide in which the polypeptide is separated from some ofthe cellular components of the cells in which it is naturally orrecombinantly produced. In one embodiment, the language “substantiallyfree of cellular material” includes preparations of an IMI polypeptidehaving less than about 30% (by dry weight) of non-IMI material (alsoreferred to herein as a “contaminating polypeptide”), more preferablyless than about 20% of non-IMI material, still more preferably less thanabout 10% of non-IMI material, and most preferably less than about 5%non-IMI material.

When the IMI polypeptide, or biologically active portion thereof, isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,more preferably less than about 10%, and most preferably less than about5% of the volume of the polypeptide preparation. The language“substantially free of chemical precursors or other chemicals” includespreparations of IMI polypeptide in which the polypeptide is separatedfrom chemical precursors or other chemicals that are involved in thesynthesis of the polypeptide. In one embodiment, the language“substantially free of chemical precursors or other chemicals” includespreparations of an IMI polypeptide having less than about 30% (by dryweight) of chemical precursors or non-IMI chemicals, more preferablyless than about 20% chemical precursors or non-IMI chemicals, still morepreferably less than about 10% chemical precursors or non-IMI chemicals,and most preferably less than about 5% chemical precursors or non-IMIchemicals. In preferred embodiments, isolated polypeptides, orbiologically active portions thereof, lack contaminating polypeptidesfrom the same organism from which the IMI polypeptide is derived.Typically, such polypeptides are produced by recombinant expression of,for example, a Triticum aestivum IMI polypeptide in plants other thanTriticum aestivum or microorganisms such as C. glutamicum, ciliates,algae or fungi.

The IMI polynucleotide and polypeptide sequences of the invention have avariety of uses. The nucleic acid and amino acid sequences of thepresent invention can be used to transform plants, thereby modulatingthe plant's resistance to imidazolinone herbicides. Accordingly, theinvention provides a method of producing a transgenic plant havingincreased tolerance to an imidazolinone herbicide comprising, (a)transforming a plant cell with one or more expression vectors comprisingone or more IMI nucleic acids, and (b) generating from the plant cell atransgenic plant with an increased resistance to an imidazolinoneherbicide as compared to a wild type variety of the plant. In oneembodiment, the multiple IMI nucleic acids are derived from differentgenomes. Also included in the present invention are methods of producinga transgenic plant having increased tolerance to an imidazolinoneherbicide comprising, (a) transforming a plant cell with an expressionvector comprising an IMI nucleic acid, wherein the nucleic acid is anon-Imi1 nucleic acid and (b) generating from the plant cell atransgenic plant with an increased resistance to an imidazolinoneherbicide as compared to a wild type variety of the plant.

The present invention includes methods of modifying a plant's toleranceto an imidazolinone herbicide comprising modifying the expression of oneor more IMI nucleic acids. Preferably, the nucleic acids are located onor derived from different genomes. The plant's resistance to theimidazolinone herbicide can be increased or decreased as achieved byincreasing or decreasing the expression of an IMI polynucleotide,respectively. Preferably, the plant's resistance to the imidazolinoneherbicide is increased by increasing expression of an IMIpolynucleotide. Expression of an IMI polynucleotide can be modified byany method known to those of skill in the art. The methods of increasingexpression of IMI polynucleotides can be used wherein the plant iseither transgenic or not transgenic. In cases when the plant istransgenic, the plant can be transformed with a vector containing any ofthe above described IMI coding nucleic acids, or the plant can betransformed with a promoter that directs expression of endogenous IMIpolynucleotides in the plant, for example. The invention provides thatsuch a promoter can be tissue specific or developmentally regulated.Alternatively, non-transgenic plants can have endogenous IMIpolynucleotide expression modified by inducing a native promoter. Theexpression of polynucleotides comprising SEQ ID NO:1 or SEQ ID NO:3 intarget plants can be accomplished by, but is not limited to, one of thefollowing examples: (a) constitutive promoter, (b) chemical-inducedpromoter, and (c) engineered promoter over-expression with for examplezinc-finger derived transcription factors (Greisman and Pabo, 1997Science 275:657).

In a preferred embodiment, transcription of the IMI polynucleotide ismodulated using zinc-finger derived transcription factors (ZFPs) asdescribed in Greisman and Pabo, 1997 Science 275:657 and manufactured bySangamo Biosciences, Inc. These ZFPs comprise both a DNA recognitiondomain and a functional domain that causes activation or repression of atarget nucleic acid such as an IMI nucleic acid. Therefore, activatingand repressing ZFPs can be created that specifically recognize the IMIpolynucleotide promoters described above and used to increase ordecrease IMI polynucleotide expression in a plant, thereby modulatingthe herbicide resistance of the plant.

As described in more detail above, the plants produced by the methods ofthe present invention can be monocots or dicots. The plants can beselected from maize, wheat, rye, oat, triticale, rice, barley, soybean,peanut, cotton, rapeseed, canola, manihot, pepper, sunflower, tagetes,solanaceous plants, potato, tobacco, eggplant, tomato, Vicia species,pea, alfalfa, coffee, cacao, tea, Salix species, oil palm, coconut,perennial grass and forage crops, for example. Forage crops include, butare not limited to, Wheatgrass, Canarygrass, Bromegrass, Wildrye Grass,Bluegrass, Orchardgrass, Alfalfa, Salfoin, Birdsfoot Trefoil, AlsikeClover, Red Clover and Sweet Clover. In a preferred embodiment, theplant is a wheat plant. In each of the methods described above, theplant cell includes, but is not limited to, a protoplast, gameteproducing cell, and a cell that regenerates into a whole plant. As usedherein, the term “transgenic” refers to any plant, plant cell, callus,plant tissue or plant part, that contains all or part of at least onerecombinant polynucleotide. In many cases, all or part of therecombinant polynucleotide is stably integrated into a chromosome orstable extra-chromosomal element, so that it is passed on to successivegenerations.

As described above, the present invention teaches compositions andmethods for increasing the imidazolinone resistance of a wheat plant orseed as compared to a wild-type variety of the plant or seed. In apreferred embodiment, the imidazolinone resistance of a wheat plant orseed is increased such that the plant or seed can withstand animidazolinone herbicide application of preferably approximately 10-400 gai ha⁻¹, more preferably 20-160 g ai ha⁻¹, and most preferably 40-80 gai ha⁻¹. As used herein, to “withstand” an imidazolinone herbicideapplication means that the plant is either not killed or not injured bysuch application.

Additionally provided herein is a method of controlling weeds within thevicinity of a wheat plant, comprising applying an imidazolinoneherbicide to the weeds and to the wheat plant, wherein the wheat planthas increased resistance to the imidazolinone herbicide as compared to awild type variety of the wheat plant, and wherein the plant comprisesone or more IMI nucleic acids. In one embodiment, the plant comprisesmultiple IMI nucleic acids located on or derived from different genomes.In another embodiment, the plant comprises a non-Imi1 nucleic acid. Byproviding for wheat plants having increased resistance to imidazolinone,a wide variety of formulations can be employed for protecting wheatplants from weeds, so as to enhance plant growth and reduce competitionfor nutrients. An imidazolinone herbicide can be used by itself forpre-emergence, post-emergence, pre-planting and at-planting control ofweeds in areas surrounding the wheat plants described herein or animidazolinone herbicide formulation can be used that contains otheradditives. The imidazolinone herbicide can also be used as a seedtreatment. Additives found in an imidazolinone herbicide formulationinclude other herbicides, detergents, adjuvants, spreading agents,sticking agents, stabilizing agents, or the like. The imidazolinoneherbicide formulation can be a wet or dry preparation and can include,but is not limited to, flowable powders, emulsifiable concentrates andliquid concentrates. The imidazolinone herbicide and herbicideformulations can be applied in accordance with conventional methods, forexample, by spraying, irrigation, dusting, or the like.

Throughout this application, various publications are referenced. Thedisclosures of all of these publications and those references citedwithin those publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art to which this invention pertains.

It should also be understood that the foregoing relates to preferredembodiments of the present invention and that numerous changes may bemade therein without departing from the scope of the invention. Theinvention is further illustrated by the following examples, which arenot to be construed in any way as imposing limitations upon the scopethereof. On the contrary, it is to be clearly understood that resort maybe had to various other embodiments, modifications, and equivalentsthereof, which, after reading the description herein, may suggestthemselves to those skilled in the art without departing from the spiritof the present invention and/or the scope of the appended claims.

EXAMPLES Example 1 Mutagenesis and Selection of Resistant Wheat Lines

Approximately 40,000 seeds of Triticum aestivum L. cv CDC Teal (Hughesand Hucl, 1993 Can. J. Plant Sci. 73:193-197) were mutagenized usingmodified procedures described by Washington and Sears (1970). Seeds werepre-soaked in distilled water for four hours, followed by treatment with0.3% EMS for six hours. Seeds were rinsed continually with tap water forseven hours and allowed to dry for approximately four hours before beingplanted in the field. The M₁ plants were selfed and the seed washarvested in bulk. Approximately 2×10⁶ M₂ plants were grown in the fieldthe following year and were sprayed at the two leaf stage with imazamoxat a rate of 40 g ai ha⁻¹ in a spray volume of 100 L ha⁻¹. Merge 0.05%(v/v) adjuvant was added to the spray solution. Six lines resistant toimazamox were selected and designated as lines 1A, 9A, 10A, 11A, 15A,and 16A. The M₃ and M₄ generations were grown in a walk-in growthchamber and plants resistant to imazamox were selected using rates of 20g ai ha⁻¹. Resistant plants were selected in the M₅ generation afterapplication of 40 g ai ha⁻¹ in the field. M₅ seed was homozygous for thetrait, as progeny testing detected no segregation for resistance toimazamox.

Example 2 Methods Used to Determine Inheritance and Allelism of IMIGenes

To determine the genetic control of resistance to imazamox in the sixwheat lines, reciprocal crosses between the six homozygous resistant M₆lines and CDC Teal (susceptible to imazamox) were made. Randomlyselected F₁ plants from each of the crosses were backcrossed to CDC Tealto form backcross (BC)F₁ populations. To investigate allelism, allpossible inter-crosses between the six mutants and SWP965001(Grandin/3*Fidel—FS-4) were made. SWP965001 is a spring wheat line thatis homozygous for the FS-4 allele. Parental genotypes were grown in awalk-in growth chamber with a 16 hour photoperiod and a 24° C. day and16° C. night temperature regime. Spikes that were ¾ emerged from theboot were emasculated and then pollinated 2-3 days after theemasculation date. Randomly selected F₂ plants from all segregatingcrosses were selfed to produce F_(2:3) families. Parental, F₁, BCF₁, F₂plants and F_(2:3) families were tested for reaction to imazamox. Allexperiments were conducted in a walk-in growth chamber with a 16 hourphotoperiod and a 23° C. day and 16° C. night temperature regime. Acompletely random design was used for all experiments. In experimentsinvolving F_(2:3) families, effort was taken to randomize both withinand among families. The F₁ and F₂ populations were screened in the sameexperiment along with parental genotypes and CDC Teal as controls. Boththe BCF₁ and F_(2:3) populations were screened in two separateexperiments along with appropriate parental genotypes as controls.

Herbicide treatments were applied to plants growing in 8×16 cell flatsat the two leaf stage using a traveling cable sprayer calibrated tospray 100 L ha⁻¹. Imazamox was applied to plants at a rate of 20 g aiha⁻¹ using an 8001 EVS nozzle at a pressure of 275 kPa. Merge surfactant(0.05% v/v) was added to the herbicide solution prior to application.Fifteen days after herbicide application, plants were rated based onparental reactions and were considered as resistant, intermediate, orsusceptible. Resistant plants were phenotypically unaffected followingherbicide treatment whereas intermediate plants were characterized byhalted growth of the first two leaves, darkening (dark-greenpigmentation) of the leaves, and the emergence of coleoptilar tillers.Susceptible plants were characterized by failure to develop new leaves,extensive leaf chlorosis, and eventually, plant death. For Mendeliananalysis of the segregating populations, plants were scored intoresistant and susceptible categories and tested for goodness of fit tovarious 1 gene, 2 gene and 3 gene models using chi-square analysis. ForF₂ and BCF₁ plant data, intermediate reactions were included in theresistant reaction category. Yates correction for continuity was used toadjust the chi-square value when only a single degree of freedom wasused in the chi-square analysis (Steele and Torrie 1980 Principles andprocedures of statistics. McGraw-Hill, New York, N.Y. pp 633).

Example 3 Results Regarding Inheritance of IMI Genes

All resistant parents produced a similar phenotype when sprayed with 20g ai ha⁻¹ of imazamox. (FIG. 1). Reciprocal crosses between theresistant lines and the susceptible parent (CDC Teal) resulted in F₁plants that survived application of imazamox (FIG. 1), indicating thatresistance to imazamox is a nuclear and not a cytoplasmic trait. Withthe exception of cross 15A×Teal, the F₁ plants resulting from each ofthe resistant lines crossed with CDC Teal displayed an intermediatereaction (FIG. 1). Since the F₁ plants were phenotypically intermediatebetween the two parents, it was concluded that resistance to imazamox inthese lines was a partially dominant trait (FIG. 1). Genetic analysis ofresistance to imidazolinones and sulfonylureas in Arabidopsis thaliana(Haughn and Somerville, 1986 Mol. Gen. Genet. 204:430-434) Zea mays(Newhouse et al., 1991 Theor. Appl. Genet. 83:65-70), Brassica napus(Swanson et al., 1989 Theor. Appl. Gen. 78:525-530), and Glycine max(Sebastian et al., 1989 Crop Sci. 29:1403-1408) also indicated thepresence of a single, partially dominant nuclear gene.

Fourteen F₁ plants resulting from the 15A×Teal cross were rated asresistant (FIG. 1). Evaluation of F₂ populations from this crossindicated that two independently segregating loci were involved inconferring resistance in this genotype (FIG. 2). Since the F₁ wouldcarry two heterozygous resistant loci, one would expect that a resistantreaction would be observed. If each of these loci alone would conferpartial dominance, additively, two heterozygous loci would produce aresistant reaction. Swanson et al. (1989) combined two semi-dominantimidazolinone resistance alleles from Brassica napus, representing twounlinked genes, to produce a F₁ hybrid that was superior inimidazolinone resistance than either of the heterozygous lines alone.The authors concluded that resistance mechanisms are additive, and ahigher level of resistance is observed in lines carrying more than oneresistance allele.

An analysis of cytoplasmic inheritance was conducted in the F₂generation by testing homogeneity of deviations from segregation ratiosbetween the two reciprocal F₂ populations. Chi-square analysis revealedno significant deviations between reciprocal populations, confirming theabsence of cytoplasmic inheritance (FIG. 2). Since cytoplasmicinheritance was absent, data from the two reciprocal populations wascombined and a total chi-square on pooled F₂ data was calculated (FIG.2).

With the exception of Teal×15A, all F₂ populations resulting fromresistant×susceptible crosses gave a good fit to a 3:1 resistantsusceptible ratio indicating segregation of a single major gene forresistance to imazamox (FIG. 2). When F₁ plants were crossed to thesusceptible parent, resulting BCF₁ populations gave a good fit to a 1:1resistant:susceptible ratio, confirming the single locus hypothesis(FIG. 2). The F₂ population data from the cross 15A×Teal fit a 15:1resistant:susceptible ratio (P=0.08), indicating segregation of twoindependent, complementary genes (FIG. 2). The BCF₁ population gave goodfit to a 3:1 resistant:susceptible ratio with a chi-square P value of0.35, confirming the results of the F₂ (FIG. 2).

Since it is speculated from F₂ data that resistance in lines 1A, 9A,10A, 11A, and 16A are controlled by a single major gene, F_(2:3)families should segregate and fit a 1:2:1 homozygousresistance:segregating:homozygous susceptible family ratio. Evaluationof F_(2:3) families indicated that crosses Teal×1A, Teal×9A, Teal×10A,Teal×11A, and Teal×16A all fit a 1:2:1 resistant:segregating:susceptibleF_(2:3) family ratio with chi-square P values of 0.64, 0.66, 0.52, 0.40,and 0.94, respectively (FIG. 3). These results confirm the results ofthe F₂ and BCF₁ data that resistance in lines 1A, 10A, 9A, 11A, andresistance in 16A is controlled by a single major gene. This pattern ofinheritance is consistent with other findings that have reported thegenetic control of resistance to AHAS inhibitor herbicides. To date,nearly all plant mutations conferring resistance to imidazolinones showthat a single, partially dominant gene controls the resistance trait. InTriticum aestivum, Zea mays, Glycine max, Arabidopsis thaliana, andNicotiana tabacum, resistance to AHAS inhibitors is inherited as asingle partially dominant nuclear gene (Newhouse et al. 1991; Newhouseet al. 1992; Chaleff and Ray, 1984 Science 223:1148-1151; Sathasivan etal., 1991 Plant Physiol. 97:1044-1050). Plant resistance to AHASinhibitor herbicides occurs mostly because of a single point mutation tothe gene encoding the AHAS enzyme (Harms et al. 1992, Mol. Gen. Genet.233:427-435; Winder and Spalding, 1988 Mol. Gen. Genet. 238:394-399).

The F₂ data resulting from the cross Teal×15A provided a good fit to a15:1 resistant:susceptible ratio, suggesting segregation of two,independently segregating loci (FIG. 2). If this is the case, F_(2:3)families should segregate and fit a 7:8:1resistant:segregating:susceptible F_(2:3) family ratio. F_(2:3) familiesfrom the cross 15A×Teal did fit the expected 7:8:1 ratio (FIG. 3),confirming the results of the F₂ and BCF₁ populations that resistance in15A is conferred by two, independent loci. To the inventor's knowledge,this is the first reported instance were two independently segregatingimidazolinone resistant alleles were identified in a single linefollowing seed mutagenesis.

Example 4 Results Regarding Allelism of IMI genes

To determine the allelic relationships of resistance genes, all possibleintercrosses between resistant lines were evaluated. No susceptibleplants were observed in the F₂ populations resulting from theinter-crosses between lines SWP965001, 1A, 9A, 10A, 15A, and 16A (FIG.4). Since these populations were not segregating, the resistance genesin these lines are either alleles at the FS-4 locus, or are very tightlylinked. Since these populations were not segregating in the F₂generation, F_(2:3) families from these crosses were not evaluated.

All inter-crosses involving line 11A did segregate in the F₂ generation,indicating the presence of a unique resistance gene in 11A (FIG. 4). Iftwo independently segregating resistance genes are present as the resultof crossing two lines, each carrying a single resistance gene, a 15:1resistant:susceptible ratio would be expected in the F₂ generation. Inthe F₂ generation, crosses SWP965001×11A, 1A×11A, 10A×11A, and 16A×11Afit the expected 15:1 resistant:susceptible ratio suggesting independentsegregation of two major resistance genes (FIG. 4). F_(2:3) familyratios from these crosses also gave a good fit to a 7:8:1resistant:segregating:susceptible ratio, confirming the results obtainedin the F₂ generation (FIG. 5). Cross 11A×9A did produce a segregating F₂population, but the ratio did not fit a 15:1 segregation ratio due to anexcess of susceptible segregants. Various other two gene hypotheses weretested, but all were found to be highly significant (Data not shown).Evaluation of F_(2:3) families from this cross, however did give goodfit to a 7:8:1 segregation ratio, indicating segregation of twoindependent genes (FIG. 5). These results confirm that the resistancegene in 11A is unique from those in lines SWP96001, 1A, 9A, 10A, and16A.

Cross 11A×15A did produce a segregating F₂ population. Since 15A iscarrying two resistance genes, one allelic to FS-4, a segregating F₂population in cross 11A×15A would indicate the presence of threesegregating genes. Segregating generations resulting from cross 15A×11Awere tested for segregation of three independent loci. F₂ plants did fitthe expected 63:1 resistant:susceptible ratio, indicating thesegregation of three independent loci (FIG. 4). These results suggestthat the second mutation in 15A is not allelic to the resistance gene in11A. F_(2:3) families were not screened as over 330 plants within eachfamily would have to be screened in order to ensure an adequate power oftest (Hanson, 1959 Agron. J. 51:711-716).

Three independent resistance loci have been identified, each with anallele conferring resistance to imazamox. Recommended rules for genelocus and allele symbolization have been published (McIntosh et al.,1998 Catalogue of Gene Symbols. Volume 5, Proceedings of the 9^(th)International Wheat Genetics Symposium. Saskatoon, Saskatchewan).Non-allelic gene loci of an enzyme that catalyze the same reactionshould be given the same symbol, corresponding to the trivial name ofthe enzyme. The trivial name for AHAS is ALS. Absent data to assign theloci to specific chromosomes and genomes, they should be designated insequential series. The designation of the phenotype observed whenchanges occur in the gene resulting in a new allele should reflect thatphenotype. Thus, it is proposed that the FS-4 imidazolinone resistanceallele be designated as Imi1 and the locus it is at designated as Als1.Imi stands for imidazolinone resistance. This designation indicates thatthe gene is a dominant trait and it is the first allele identified.Segregating F₂ and F_(2:3) population data suggests that 15A and 11Acarry two new independent resistance alleles at different loci (FIGS. 2and 3). The designations for these alleles are Imi2 for the 11A mutationat the Als2 locus and Imi3 for the second 15A mutation at the Als3locus.

Identified herein are three independently segregating alleles conferringresistance to imazamox, namely Imi1 (1A, 9A, 10A, 15A and 16A), Imi2(11A), and Imi3 (15A). It is proposed that each of the three identifiedalleles are associated with a different structural gene coding forherbicide-insensitive forms of AHAS. Since wheat is a hexaploid,multiple AHAS loci would be expected. Other polyploid species have beenfound to have more than one copy of AHAS. In Nicotiana tabacum, anallotetraploid, two AHAS genes have been identified and characterized(Mazur et al. 1987). Chaleff and Ray (1984) identified two independentlysegregating sulfonylurea resistance alleles in Nicotiana tabacum, eachcoding for an altered form of AHAS. Zea mays possesses twoconstitutively expressed identical AHAS genes (Fang et al., 1992 PlantMol. Biol. 18:1185-1187). In allotetraploid Brassica napus and Gossypiumhirsutum, an AHAS multi-gene family consisting of five and six members,respectively, is present (Rutledge et al., 1991 Mol. Gen. Genet.229:31-40; Grula et al., 1995 Plant Mol. Biol. 28:837-846). Higherlevels of resistance to herbicides have been observed in polyploidspecies when multiple resistance alleles are present. Swanson et al.(1989 Theor. Appl. Gen. 78:525-530) combined two unique imidazolinoneresistance alleles from two homozygous Brassica napus lines resulting inprogeny with a higher level of resistance than either homozygous linealone. Creason and Chaleff (1988 Theor. Appl. Genet. 76:177-182)identified Nicotiana tabacum plants homozygous for two mutations thatconferred resistance to sulfonylureas. Plants homozygous for bothmutations were five-fold more resistant to foliar applications ofchlorsulfuron than were plants homozygous for each single mutation. Thepresent invention proposes producing increased levels of resistance toan imidazolinone herbicide in wheat by combining any two or all threeresistance alleles.

Example 5 Tolerance to Imidazolinone Herbicides in Teal11A, Teal15A andTeal11A/15A Hybrid

The increased tolerance exhibited by Teal 11A and Teal 15A to 20 gramsper hectare of imazamox has been exemplified in previous examples by theability to distinguish tolerant from susceptible parental and segregantplants in inheritance studies. Teal 11A has been shown to confer similarlevels of tolerance to imidazolinone herbicides to that conferred by theFS4 mutation in Fidel in various greenhouse and field comparisons. Thesimilarity in tolerance is also reflected in comparing the in vitroactivity of AHAS extracted from tolerant plants. This is possiblebecause the tolerance in Teal 11A, Teal 15A, and FS4 is due to amutation in the AHAS enzyme rendering it resistant to inhibition byimidazolinone herbicides. FIG. 6 indicates that the activity of AHASenzyme extracted from Teal 11A and BW755, a line containing FS4, changessimilarly as the rate of imazamox increases, and both have a higherpercentage of active (resistant) enzyme at the highest concentration ofimazamox than does the wild type check, Teal.

The presence of two IMI nucleic acids in Teal 15A provides increasedtolerance to imidazolinone herbicides compared to a line such as BW755carrying only one IMI nucleic acid. This increased tolerance isreflected both in less injury at higher herbicide rates, but in havingmore uninhibited AHAS enzyme activity. FIG. 7 illustrates that a 10×rate of imazamox (200 g/ha), all treated one gene plants were injured,while no two gene plants were injured. At all concentrations of imazamoxin an in vitro assay of AHAS activity (FIG. 6), but particularly at thehighest concentrations, Teal ISA had a higher percentage of active(resistant) enzyme than did either of the single gene lines, Teal11A andBW755.

Combining three non-allelic genes each conferring tolerance toimidazolinone herbicides results in greater tolerance than with only twonon-allelic genes (FIG. 7). At a 30× rate, or 600 g/ha of imazamox, overhalf of plants sustained no injury in a still-segregating selfedpopulation of Teal15A crossed with Teal 11A, while all plants of thehomozygous population of Teal15A sustained injury.

1. A method of controlling weeds within the vicinity of a wheat plant,comprising applying an imidazolinone herbicide to the weeds and to thewheat plant, wherein the wheat plant has increased resistance to theimidazolinone herbicide as compared to a wild type variety of the wheatplant, wherein the plant comprises multiple IMI nucleic acids, andwherein the nucleic acids are from different genomes.
 2. The method ofclaim 1, wherein the multiple IMI nucleic acids are selected from thegroup consisting of an Imi1 nucleic acid, an Imi2 nucleic acid and anImi3 nucleic acid.
 3. The method of claim 1, wherein the plant comprisesan Imi1 nucleic acid and an Imi2 nucleic acid.
 4. The method of claim 1,wherein the plant comprises an Imi1 nucleic acid and an Imi3 nucleicacid.
 5. The method of claim 1, wherein at least one of the multiplenucleic acids is selected from the group consisting of: a) apolynucleotide comprising the polynucletide sequence set forth in SEQ IDNO: 1; b) a polynucleotide comprising the polynucletide sequence setforth in SEQ ID NO: 3; c) a polynucleotide encoding a polypeptidecomprising the amino acid sequence set forth in SEQ ID NO:2; d) apolynucleotide encoding a polypeptide comprising the amino acid sequenceset forth in SEQ ID NO:4; and e) a polynucleotide encoding an IMIprotein comprising at least 90% amino acid sequence identity to theamino acid sequence set forth in at least one of SEQ ID NOS: 2 and 4,wherein the polypeptide comprises a serine to asparagine substitution inDomain E.
 6. The method of claim 1, wherein the imidazolinone herbicideis selected from the group consisting of2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-nicotinic acid,2-(4-isopropyl)-4-methyl-5-oxo-2-imidazolin-2-yl)-3-quinolinecarboxylicacid, 5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-nicotinicacid,2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-(methoxymethyl)-nicotinicacid, 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-methylnicotinicacid, and a mixture of methyl6-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-m-toluate and methyl2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-p-toluate.
 7. Themethod of claim 1, wherein the imidazolinone herbicide is5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-nicotinic acid.8. The method of claim 1, wherein the imidazolinone herbicide is2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-(methoxymethyl)-nicotinicacid.
 9. A method of controlling weeds within the vicinity of a wheatplant, comprising applying an imidazolinone herbicide to the weeds andto the wheat plant, wherein the wheat plant has increased resistance tothe imidazolinone herbicide as compared to a wild type variety of thewheat plant, and wherein the plant comprises an IMI nucleic acid that isa non-Imi1 nucleic acid.
 10. The method of claim 9, wherein the IMInucleic acid is selected from the group consisting of an Imi2 nucleicacid and an Imi3 nucleic acid.
 11. The method of claim 9, wherein theIMI nucleic acid is selected from the group consisting of: a) apolynucleotide comprising the polynucletide sequence set forth in SEQ IDNO: 3; b) a polynucleotide encoding a polypeptide comprising the aminoacid sequence set forth in SEQ ID NO:4; and c) a polynucleotide encodingan IMI protein comprising at least 90% amino acid sequence identity tothe amino acid sequence set forth in SEQ ID NO: 2, wherein thepolypeptide comprises a serine to asparagine substitution in Domain E.12. The method of claim 9, wherein the imidazolinone herbicide isselected from the group consisting of2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-nicotinic acid,2-(4-isopropyl)-4-methyl-5-oxo-2-imidazolin-2-yl)-3-quinolinecarboxylicacid, 5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-nicotinicacid,2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-(methoxymethyl)-nicotinicacid, 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-methylnicotinicacid, and a mixture of methyl6-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-m-toluate and methyl2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-p-toluate.
 13. Themethod of claim 9, wherein the imidazolinone herbicide is5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-nicotinic acid.14. The method of claim 9, wherein the imidazolinone herbicide is2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-(methoxymethyl)-nicotinicacid.